APC promoter 1B deletion in familial polyposis--implications for mutation-negative families.

Over 1500 adenomatous polyposis coli (APC) gene mutations have already been identified as causative of familial adenomatous polyposis (FAP). However, routine genetic testing fails to detect mutations in about 10% of classic FAP cases. Recently, it has been shown that a proportion of mutation-negative FAP cases bear molecular changes in deep intronic and regulatory sequences. In this study, we used direct sequencing, followed by multiplex ligation-dependent probe amplification (MLPA) of genomic DNA from family members, affected by classic FAP. We first reported the family as mutation negative. With the launch of a new version of MLPA kit, we retested the family and a novel full deletion of promoter 1B was detected. The exact breakpoints of the deletion were determined by array comparative genomic hybridization (CGH) and long range polymerase chain reaction (PCR), followed by direct sequencing. The total APC expression levels were investigated by quantitative polymerase chain reaction (qPCR) assay and allele-specific expression (ASE) analysis. The APC gene expression was highly reduced, which indicates causative relationship. We suggest that there is a significant possibility that APC promoter 1B mutations could be found in mutation-negative FAP patients. In the light of our findings it seems reasonable to consider targeted genetic re-analysis of APC promoter 1B region in a larger cohort of unsolved cases.

Expression profile of Rho kinases in urinary bladder cancer.

PURPOSE: To investigate the expression of RhoA, RhoB, RhoC, Rac1 and Cdc42 kinases in urothelial cell carcinoma (UCC) of the urinary bladder and determine the expression profile of 107 Rho-associated genes, including GTPases, GDIs, GAPs and GEFs. METHODS: Rho expression was investigated using microarrays, qPCR and Western blotting in 77 UCC specimens with paired normal urothelium. Computational analysis was also performed on Gene Expression Omnibus datasets. Further microarray analysis was carried out for the expression profiling of the Rho-associated genes. RESULTS: RhoB mRNA and protein levels were significantly lower in UCC, suggesting a tumour-suppressor role. On the contrary, mRNA of RhoC and protein levels of RhoA, RhoC and Cdc42, respectively, were significantly higher in UCC vs. normal tissue. High Cdc42 mRNA levels correlated with worse overall survival (p=0.027), whereas high RhoB mRNA levels correlated both with better overall (p=0.0258) and cancer-specific (p=0.0272) survival. Computational analysis verified the expression profile of Rho kinases among superficial UCCs, muscle-invasive UCCs and normal tissues. CONCLUSION: The majority of the Rho-related genes showed over-expression in UCC vs. normal tissue. Alterations in RhoA, RhoB, RhoC, Rac1 and Cdc42 expression play a significant role in the genesis and progression of UCC of the urinary bladder.

Microsatellite instability and promoter hypermethylation of MLH1 and MSH2 in patients with sporadic colorectal cancer.

PURPOSE: Inactivation of the genes involved in DNA mismatch repair (MMR) is associated with microsatellite instability (MSI) and loss of heterozygosity (LOH). The aim of the current study was to assess the presence of MSI and promoter hypermethylation of MLH1 and MSH2 in Bulgarian PATIENTS WITH SPORADIC COLORECTAL CANCER (CRC) AND TO ANALYZE THEIR POSSIBLE EFFECT ON THE DEVELOPMENT, PROGRESSION AND PROGNOSIS OF THE DISEASE. METHODS: We examined MSI in 126 patients with sporadic CRC and the methylation status of the MLH1 and MSH2 promoter regions in the cases with MSI/LOH by using a panel of 5 microsatellite markers (BAT26, D5S346, D18S35, D2S123 and FGA) and methyl-specific PCR (MSP) of bisulfite converted DNA. RESULTS: MSI/LOH was found in 36 (28.6%) patients. Among them, 30 were analyzed for promoter hypermethylation of MLH1 and we detected hypermethylation in 15 (50%) of them, whereas promoter hypermethylation of MSH2 was observed only in one case. The presence of MSI/LOH was associated with younger age (p=0.002), more advanced stage (III/IV stage) (p=0.029), lower degree of differentiation (p=0.001), and right-sided tumor localization (p=0.0002), but not with overall survival (log rank, p=0.566). CONCLUSION: Our data suggest that sporadic CRCs with MSI/LOH are more aggressive, develop earlier and progress faster to more advanced stage. The most frequent cause of failure of DNA MMR system appeared to be the hypermethylation of CpG islands of the promoter region of MLH1, whereas the methylation of MSH2 was a rare event.

CHEK2 I157T and colorectal cancer in Bulgaria.

PURPOSE: Germline variants of the CHEK2 gene have been shown to act as low-penetrance cancer susceptibility alleles for a wide range of human malignancies. CHEK2 I157T has particularly been linked to colorectal cancer (CRC) risk. We aimed at establishing the population frequency and contribution of this variant to colorectal carcinogenesis in Bulgaria. METHODS: We have genotyped 802 population controls and 343 CRC patients from Bulgaria for the CHEK2 I157T variant. RESULTS: Heterozygous were 9 of 343 patients (2.62%, odds ratio/OR=1.0, 95% confidence interval/CI = 0.42 - 2.33, p=0.99% and 21 of 802 controls (2.62%). Higher frequencies were found among patients with multiple polyposis (2/40, 5%, p=0.28) and the rarer mucinous histology (1/11, 9.09%, p= 0.26). CONCLUSION: We conclude that CHEK2 I157T is not relevant for CRC risk in Bulgaria, but studies on a larger scale might help evaluate its possible significance in respect to disease characteristics.

Microsatellite instability in Bulgarian patients with endometrial cancer.

PURPOSE: Microsatellite instability (MSI) is a frequent event in different types of cancer. In several studies MSI was shown to have both clinical and prognostic value. The aim of our study was to determine the frequency of MSI in Bulgarian patients with endometrial cancer (EC) and the possible relation of this phenomenon to their clinicopathological characteristics. PATIENTS AND METHODS: A total of 33 histologically confirmed EC patients were analyzed for tumor MSI using a panel of 6 microsatellite markers. RESULTS: We identified MSI in 30% of endometrial cancer cases. Six of them had high degree of MSI (MSI-H), and 4 displayed low degree of MSI (MSI-L). CONCLUSION: The frequency of MSI in Bulgarian EC patients does not differ significantly from that reported in other European studies.

A case of exocrine-endocrine neoplasm of the pancreas in a patient with ulcerative colitis with literature review.

Until now only Japanese authors have reported 4 cases of pancreatic neoplasm associated with ulcerative colitis (UC). We report on a case of a 44-year-old woman who was operated on for complicated UC and an exocrine-endocrine neoplasm of the pancreas, where the endocrine component was presented by pancreatic polypeptide (PP)-producing cells. By means of molecular genetics methods we found microsatellite instability (MSI) in the markers D18S35, FGA and p53 in the colonic lining, and loss of heterozygosity (LOH) in the p53 marker in the pancreatic tumor. A literature review concerning the coexistence of these two conditions showed that PP is involved in the pathogenesis of the UC and that UC increases the risk of development of extracolonic neoplasms.